1. Technical Field
The invention relates to methods and materials involved in the production of organic products.
2. Background Information
Organic products such as lactic acid have many important industrial uses. For example, organic acids can be used to synthesize plastic materials as well as other products. To meet the increasing need for organic products, more efficient and cost effective production methods are being developed. One such method involves the use of bacteria. Specifically, certain bacteria can produce large quantities of particular organic products under certain fermentation conditions. The use of living bacteria as factories, however, is limited by the inability of the bacteria to grow as the organic product accumulates in the growth media. To circumvent such limitations, various product purification techniques have been employed during product synthesis. In addition, the use of microorganisms other than bacteria has been attempted. In fact, Saccharomyces cerevisiae, which is known to be acid tolerant, has been genetically modified in an attempt to produce lactic acid. Specifically, S. cerevisiae cells were modified by providing the cells with a bovine lactate dehydrogenase cDNA and disrupting endogenous pyruvate decarboxylase genes (PDC1, PDC5, and PDC6). While these modified S. cerevisiae cells produced some lactic acid, cell growth was suppressed leading to the conclusion that both cell growth and lactic acid production need improvement.
The present invention relates generally to methods and materials for producing organic products. Specifically, the invention provides yeast cells, methods for culturing yeast cells, methods for making yeast cells, nucleic acid constructs, and methods and materials for producing various organic products. The invention is based on the discovery that particular microorganisms (e.g., bacterial and fungal microorganisms) can be genetically manipulated such that they have the ability, under specific culture conditions, to grow, utilize various carbon sources for growth as well as product production, and produce a desired organic product for commercial purposes. For example, the yeast cells provided herein can grow and produce an organic product when cultured at low pH and high temperature. Having the ability to grow rapidly and produce an organic product efficiently under, for example, low pH and high temperature conditions is particularly advantageous. Specifically, the ability of a microorganism to tolerate low pH obviates the need to maintain a neutral pH environment, which can be difficult and expensive during large-scale production processes. In addition, the methods and materials needed to recover the desired organic product from a low pH broth can be more practical and efficient than those required to recover the same organic product from a broth having a more neutral pH. For example, certain organic acid products can precipitate out of solution as the pH drops below the product""s pKa value, making recovery quite simple. Further, the ability of a microorganism to tolerate high temperatures obviates the need to maintain cool temperatures during the growth and production phases. Clearly, reducing the need to lower the temperature in a large volume tank of broth during large-scale production processes makes the overall process more efficient and less expensive. Moreover, the ability of a microorganism to tolerate both low pH and high temperature provides a convenient method for preventing contamination by other less tolerant microorganisms during the large-scale production processes.
It is important to note that a critical aspect relating to the ability to produce a desired organic product for commercial purposes can be the specific productivity at which that desired organic product is produced. For example, providing a high specific productivity using the methods and materials as described herein can allow a microorganism to generate the energy needed for cell maintenance when exposed to culture conditions such as low pH and high temperature. This required energy can be generated via a fermentation pathway under substantially anaerobic conditions, rather than relying on the generation of energy via the respiratory pathway. Obtaining energy via a fermentation pathway is particularly advantageous when producing an organic product that does not require the respiratory pathway since essentially all of the provided carbon source can be used to produce the desired organic product.
The invention also is based on the discovery that the utilization of a carbon source by certain genetically manipulated microorganisms can be controlled and directed predominately towards the production of either biomass or a desired organic product. In general terms, the invention involves two types of culturing processes. One culturing process involves culturing microorganisms under specific culture conditions, depending on the microorganism and desired outcome, that promote biomass production, while the other involves a different set of culture conditions, also dependent upon the microorganism and desired outcome, that promotes the production of a desired organic product. Clearly, having the ability to manipulate the utilization of a carbon source during large-scale production processes provides manufacturers with greater flexibility and more control than is otherwise possible.
In addition, the invention is based on the discovery that certain microorganisms can be genetically manipulated such that most, if not all, of a carbon source is utilized for the production of either biomass or a desired organic product. Specifically, the invention provides yeast cells that are modified such that biosynthesis pathways that divert the utilization of a carbon source away from the production of biomass or the desired organic product are inactivated. Inactivating such biosynthesis pathways provides microorganisms that can efficiently grow and produce the desired product.
In general, the invention features a yeast cell containing an exogenous nucleic acid molecule, with the exogenous nucleic acid molecule encoding a polypeptide having enzymatic activity within the cell. The nucleic acid can be incorporated into the genome of the cell. The enzymatic activity leads to the formation of an organic product which, in some embodiments, is secreted from the cell. The cell further has a crabtree-negative phenotype and produces the organic product. The cell can be, for example, from the genus Kluyveromyces, Pichia, Hansenula, Candida, Trichosporon, or Yamadazyma. The organic product can be, for example, a fermentation product, a pyruvate-derived product, an organic acid, or a carboxylate such as lactate. In one embodiment, the polypeptide can have lactate dehydrogenase activity. For example, the exogenous nucleic acid can encode a bacterial lactate dehydrogenase or fungal lactate dehydrogenase such as a K. lactis fungal lactate dehydrogenase.
In another embodiment, the cell contains four exogenous nucleic acid molecules, each of the four exogenous nucleic acid molecules encoding a different polypeptide. For example, the first of the four exogenous nucleic acid molecules can encode a first polypeptide having lactate dehydrogenase activity, the second can encode a second polypeptide having CoA-transferase activity, the third can encode a third polypeptide having lactyl-CoA dehydratase activity, and the fourth can encode a fourth polypeptide having acrylyl-CoA hydratase activity. Such a cell can produce acrylate as the carboxylate product. Alternatively, the first of the four exogenous nucleic acid molecules can encode a first polypeptide having 2-dehydro-3-deoxy-D-pentanoate aldolase activity, the second can encode a second polypeptide having xylonate dehydratase activity, the third can encode a third polypeptide having xylonolactonase activity, and the fourth can encode a fourth polypeptide having D-xylose dehydrogenase activity. Such a cell can produce a carbohydrate, such as D-xylose, as the organic product.
In yet another embodiment, the cell contains six exogenous nucleic acid molecules, each of the six exogenous nucleic acid molecules encoding a different polypeptide. For example, the first of the six exogenous nucleic acid molecules can encode a first polypeptide having 2,5-dioxovalerate hehydrogenase activity, the second can encode a second polypeptide having 5-dehydro-4-deoxy-D-glucarate dehydrogenase activity, the third can encode a third polypeptide having glucarate dehydratase activity, the fourth can encode a fourth polypeptide having aldehyde dehydrogenase activity, the fifth can encode a fifth polypeptide having glucuronolactone reductase activity, and the sixth can encode a sixth polypeptide having L-gulonolactone oxidase activity. Such a cell can produce a vitamin, for example L-ascorbate, as the organic product.
The organic product can contain more than three carbon atoms, and can be, for example, an amino acid.
In another embodiment, the cell is able to catabolize a pentose carbon such as ribose, arabinose, xylose, and lyxose.
In another embodiment the cell has reduced pyruvate decarboxylase activity or reduced alcohol dehydrogenase activity. For example, the cell can lack all pyruvate decarboxylase activity. The reduced pyruvate decarboxylase activity can be due to a disrupted genetic locus, where the locus normally has the nucleic acid sequence that encodes pyruvate decarboxylase. Alternatively, the cell could contain an antisense molecule, such as a ribozyme, that corresponds to an endogenous nucleic acid sequence, where the antisense molecule reduces the pyruvate decarboxylase activity. The cell can also contain an additional exogenous nucleic acid molecule that functions as a killer plasmid.
In another embodiment, the enzymatic activity of the polypeptide encoded by the exogenous nucleic acid leads to the formation of the organic product in an NADH-consuming manner.
In another embodiment, the cell produces at least about 60 grams of the organic product for every 100 grams of glucose consumed when the cell is cultured under optimal conditions for the production of the organic product.
In another aspect, the invention features a cell, e.g., a yeast cell, that contains an exogenous nucleic acid molecule, where the exogenous nucleic acid molecule encodes a polypeptide that promotes catabolism of a pentose carbon by the cell. The polypeptide can be, for example, xylose reductase, xylitol dehydrogenase, or xylulokinase, and the pentose carbon can be, for example, ribose, arabinose, xylose, and lyxose. The cell can further catabolize a hexose carbon and can, if desired, simultaneously catabolize the hexose carbon and the pentose carbon. The hexose carbon can be, for example, allose, altrose, glucose, mannose, gulose, iodose, fructose, galactose, and talose.
In another aspect, the invention features a yeast cell containing an exogenous nucleic acid molecule, where the exogenous nucleic acid molecule encodes a polypeptide that promotes accumulation of acetyl-CoA in the cytoplasm of the cell. The polypeptide can be a polypeptide that has citrate lyase activity, or can be a mitochondrial membrane polypeptide that promotes acetyl-CoA permeability across the mitochondrial membrane. The cell can have reduced pyruvate decarboxylase activity or reduced alcohol dehydrogenase activity. Alternatively, the yeast cell can lack ethanol production, and can have a growth rate under culture conditions lacking ethanol and acetate that is greater than the growth rate observed for a comparable yeast cell lacking ethanol production.
In yet another aspect, the invention features a yeast cell having reduced activity of a mitochondrial polypeptide, where the cell has a crabtree-negative phenotype. Such a cell can be from, for example, the genus Kluyveromyces, Pichia, Hansenula, Candida, Trichosporon, or Yamadazyma. The cell can completely lack the activity. The cell can contain a disrupted locus, where the locus normally includes a nucleic acid sequence that encodes the mitochondrial polypeptide. The mitochondrial polypeptide can be a Krebs cycle enzyme. Further, the cell can accumulate a Krebs cycle product. The cell can include an exogenous nucleic acid molecule, where the exogenous nucleic acid molecule encodes a polypeptide having enzymatic activity within the cell, with the enzymatic activity leading to formation of an organic product, such that the cell produces the organic product. The organic product can be, for example, citrate, xcex1-ketoglutarate, succinate, fumarate, malate, and oxaloacetate. The polypeptide can be a polypeptide that participates in the catabolism of lactate or acetate.
In another aspect, the invention features a method for producing an organic product. The method includes providing yeast cells, where the cells include an exogenous nucleic acid molecule that encodes a polypeptide having enzymatic activity within the cells, where the enzymatic activity leads to the formation of the organic product, and where the cells have a crabtree-negative phenotype, and culturing the cells with culture medium such that the organic product is produced. The yeast cells can be from within the genus Kluyveromyces, Pichia, Hansenula, Candida, Trichosporon, or Yamadazyma. The organic product can be a fermentation product, a pyruvate-derived product, an organic product containing more than three carbon atoms, a carboxylate, carbohydrate, amino acid, vitamin, or lipid product. The organic product further can be lactate, glycerol, acrylate, xylose, ascorbate, citrate, isocitrate, xcex1-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, or oxaloacetate. In some embodiments, the organic product is secreted by the cells. The method can result in cells having reduced pyruvate decarboxylase activity or reduced alcohol dehydrogenase activity. The enzymatic activity can lead to the formation of the organic product in an NADH-consuming manner.
Cells made by these methods can produce at least about 60 grams of the organic product for every 100 grams of glucose consumed when the culturing step is optimal for production of the organic product. The culture medium, which can be liquid, can include an inhibitor of cellular respiration, such as antimycin A, cyanide, or azide. The culturing step can include growing the cells under aerobic growth conditions followed by contacting said cells with an inhibitor of cellular respiration.
In an alternative embodiment, the culturing step includes incubating the cells under anaerobic culture conditions. In a further alternative embodiment, the culturing step includes growing the cells under aerobic growth conditions followed by incubating the cells under anaerobic culture conditions. The culturing step can also include culturing the cells at a temperature greater than about 35xc2x0 C.
In one embodiment, the culture medium has an organic pH value less than about 3.0, and/or an inorganic pH value less than about 3.0. In another embodiment, the medium contains a pentose carbon such as ribose, arabinose, xylose, or lyxose. The medium also can include a corn fiber hydrolysate having, for example, a pH value between about 2.0 and about 6.5.
In another aspect, the invention features a method for producing an organic product, the method including a) providing yeast cells containing an exogenous nucleic acid molecule encoding a polypeptide that promotes catabolism of a pentose carbon by the cell, where the cell contains an enzymatic activity that leads to the formation of said organic product, and b) culturing the cells with culture medium such that the organic product is produced.
In yet another aspect, the invention features a method for producing an organic product, the method including a) providing yeast cells, where the cells include an exogenous nucleic acid molecule encoding a polypeptide that promotes accumulation of acetyl-CoA in the cytoplasm of the cell, and where the cell contains an enzymatic activity that leads to the formation of the organic product, and b) culturing the cells with culture medium such that the organic product is produced.
In another aspect, the invention features a method for producing an organic product, the method including a) providing yeast cells having reduced activity of a mitochondrial enzyme, wherein reduction of the activity leads to the accumulation of the organic product, and b) culturing said cells with culture medium such that said organic product is produced.
In another aspect, the invention features a method for culturing yeast cells having a crabtree-negative phenotype, the method including culturing the cells with culture medium, where the culture medium has an organic pH value less than about 3.0 and/or an inorganic pH value less than about 3.0. The culturing step can include culturing the cells at a temperature greater than about 35xc2x0 C. The culture medium can include an inhibitor of cellular respiration. The culture medium also can include a pentose carbon. In another embodiment, the culture medium can include a corn fiber hydrolysate.
In another aspect, the invention features a method for culturing yeast cells having a crabtree-negative phenotype, the method including culturing the cells with culture medium, where the culture medium includes a corn fiber hydrolysate.
In another aspect, the invention features a method for culturing yeast cells having a crabtree-negative phenotype, the method including culturing the cells with culture medium at a temperature greater than about 35xc2x0 C., with the culture medium having an inorganic pH value less than about 3.0.
In another aspect, the invention features a method for culturing yeast cells having a crabtree-negative phenotype, the method including culturing the cells with culture medium at a temperature greater than about 35xc2x0 C., with the culture medium including a pentose carbon.
In another aspect, the invention features a method for culturing yeast cells having a crabtree-negative phenotype, the method including culturing the cells with culture medium at a temperature greater than about 35xc2x0 C., with the culture medium including a corn fiber hydrolysate.
In another aspect, the invention features a nucleic acid construct that includes a recombination sequence and a selected sequence, with the recombination sequence corresponding to a genomic sequence of a cell having a crabtree-negative phenotype, with the genomic sequence encoding an enzyme expressed by the cell, and with the selected sequence encoding an enzyme that leads to the formation of an organic product within the cell. The selected sequence can be within the recombination sequence such that the selected sequence is flanked on each end by the recombination sequence.
In another aspect, the invention features a method for making a recombinant yeast cell, including providing a yeast cell having a crabtree-negative phenotype, selecting an end product, identifying which exogenous enzyme or enzymes need to be added to the cell to produce the end product, identifying which endogenous enzyme or enzymes whose activity is to be reduced in said cell to allow production of said end product within said cell, adding the identified exogenous enzyme or enzymes to the provided yeast cell, and reducing the activity of the identified endogenous enzyme or enzymes in the provided yeast cell such that the cell produces the end product under culture conditions.
In another aspect, the invention features a corn fiber hydrolysate, the hydrolysate having a pH value between about 2.0 and about 6.5. The hydrolysate can include glucose, xylose, and arabinose. The hydrolysate can include about 40 grams/L glucose, about 40 grams/L xylose, and about 20 grams/L arabinose. Alternatively, the hydrolysate can include about 38.7 grams/L glucose, about 39.1 grams/L xylose, about 20.7 grams/L arabinose, and about 1.6 grams/L furfural.
In another aspect, the invention features a method for making an organic product, including a) culturing a microorganism under culture conditions, where the microorganism has reduced enzymatic activity; the enzymatic activity can be pyruvate decarboxylase, alcohol dehydrogenase, aldehyde dehydrogenase, or acetyl-CoA synthase activity; the microorganism exhibits a growth rate in the absence of ethanol and acetate that is at least about 30 percent of that observed for a corresponding microorganism not having said reduced enzymatic activity, and b) changing the culture conditions to promote production of the organic product.
In another aspect, the invention features a method for making an organic product, including a) culturing a microorganism under culture conditions that promote cellular respiration, where the microorganism has reduced enzymatic activity; the enzyme activity can be pyruvate decarboxylase, alcohol dehydrogenase, aldehyde dehydrogenase, or acetyl-CoA synthase activity, with the microorganism exhibiting a growth rate in the absence of ethanol and acetate that is at least about 30 percent of that observed for a corresponding microorganism not having such reduced enzymatic activity, and b) changing the culture conditions to reduce cellular respiration, thereby promoting production of the organic product.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.